CRISPR/Cas9 bioluminescence-based assay for monitoring CFTR trafficking to the plasma membrane.

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.

Western Blotting with Fast SDS-PAGE and Semi-Dry Protein Transfer.

Western blotting is a method widely used in cell and molecular biology for the specific detection of proteins in biological samples. It is time-consuming and normally takes up to 2 days to complete. This protocol introduces a more time-efficient method to complete western blots, covering the preparation of protein extracts (including strategies for solubilization), electrophoretic separation of proteins, transfer of proteins to the membrane, and probing with antibodies. We describe an SDS-PAGE protocol that achieves a gradient-like separation of proteins (10-400 kDa) on a single-percentage polyacrylamide gel in only 45 min. Additionally, we present a rapid (10-14 min) semi-dry transfer of proteins from standard Tris/glycine polyacrylamide gels onto a membrane using homemade Tris/HEPES- or Tris/EPPS-based buffers. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Support Protocol 1: Cell lysis and protein extraction Support Protocol 2: Protein quantification with BCA assay and sample preparation for loading on gel Basic Protocol 2: Protein transfer with a fast semi-dry transfer (FSDT) buffer Basic Protocol 3: Immunoprobing, chemiluminescent visualization, stripping, and reuse of membranes.

Treatment With LAU-7b Complements CFTR Modulator Therapy by Improving Lung Physiology and Normalizing Lipid Imbalance Associated With CF Lung Disease.

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasians, affecting more than 100,000 individuals worldwide. It is caused by pathogenic variants in the gene encoding CFTR, an anion channel at the plasma membrane of epithelial and other cells. Many CF pathogenic variants disrupt the biosynthesis and trafficking of CFTR or reduce its ion channel function. The most frequent mutation, loss of a phenylalanine at position 508 (F508del), leads to misfolding, retention in the endoplasmic reticulum, and premature degradation of the protein. The therapeutics available for treating CF lung disease include antibiotics, mucolytics, bronchodilators, physiotherapy, and most recently CFTR modulators. To date, no cure for this life shortening disease has been found. Treatment with the Triple combination drug therapy, TRIKAFTA®, is composed of three drugs: Elexacaftor (VX-445), Tezacaftor (VX-661) and Ivacaftor (VX-770). This therapy, benefits persons with CF, improving their weight, lung function, energy levels (as defined by reduced fatigue), and overall quality of life. We examined the effect of combining LAU-7b oral treatment and Triple therapy combination on lung function in a F508deltm1EUR mouse model that displays lung abnormalities relevant to human CF. We assessed lung function, lung histopathology, protein oxidation, lipid oxidation, and fatty acid and lipid profiles in F508deltm1EUR mice.

The gradient-like separation and reduced running time with Tris-Tricine-HEPES buffer for SDS-PAGE.

Tris-Glycine-SDS is the most commonly used running buffer for SDS-PAGE. Relatively long running times, poor resolution of small molecular weight proteins and excessive heat at higher voltages impede its utility for high throughput downstream applications such as western blot. Here we describe a protocol for gradient-like simultaneous separation of small (<10 kDa) and large (>400 kDa) proteins in a single percentage polyacrylamide Tris-Acetate gel using a novel running buffer composed of Tris, Tricine and HEPES.

One-Step Synthesis of Nanoliposomal Copper Diethyldithiocarbamate and Its Assessment for Cancer Therapy.

The metal complex copper diethyldithiocarbamate (CuET) induces cancer cell death by inhibiting protein degradation and induces proteotoxic stress, making CuET a promising cancer therapeutic. However, no clinical formulation of CuET exists to date as the drug is insoluble in water and exhibits poor bioavailability. To develop a scalable formulation, nanoliposomal (LP) CuET was synthesized using ethanol injection as a facile one-step method that is suitable for large-scale manufacturing. The nanoparticles are monodispersed, colloidally stable, and approximately 100 nm in diameter with an encapsulation efficiency of over 80%. LP-CuET demonstrates excellent stability in plasma, minimal size change, and little drug release after six-month storage at various temperatures. Additionally, melanoma cell lines exhibit significant sensitivity to LP-CuET and cellular uptake occurs predominantly through endocytosis in YUMM 1.7 cancer cells. Intracellular drug delivery is mediated by vesicle acidification with more nanoparticles being internalized by melanoma cells compared with RAW 264.7 macrophages. Additionally, the nanoparticles preferentially accumulate in YUMM 1.7 tumors where they induce cancer cell death in vivo. The development and characterization of a stable and scalable CuET formulation illustrated in this study fulfils the requirements needed for a potent clinical grade formulation.